During development of the nervous system growing axons (nerve fibers) are guided to their targets by responses of their motile tips (growth cones) to molecular cues in the environment. The long term goal of this work is to understand the intracellular signaling mechanisms that regulate axon outgrowth and guidance in the mammalian CNS. Wnt5a is a secreted protein that guides axons in several developing cortical pathways in vivo. In dissociated cultures Wnt5a repels growing cortical axons while increasing their rate of outgrowth. Separate plasma membrane receptors Ryk and Fz (Frizzled) and distinct calcium signaling components (IP3 receptors and TRP calcium channels) were found to differentially mediate cortical axon outgrowth and repulsive growth cone turning behaviors evoked by Wnt5a. In the first specific aim, the role of these Wnt5a signaling mechanisms will be tested in E16.5 mouse cortical slices, which preserve an in vivo like CNS organization. Confocal microscopic imaging will be carried out in the corpus callosum, a cortical pathway that links the two cerebral hemispheres and is surrounded by gradients of Wnt5a. Callosal axon growth rates will be analyzed in relation to levels of calcium activity as measured by calcium biosensors. Selective disruption of Wnt5a receptors and calcium signaling components will test their role in callosal axon outgrowth and guidance. In the second specific aim mechanisms for repulsive growth cone turning in Wnt5a gradients will be investigated with live cell imaging of embryonic mouse cortical cultures in order to relate asymmetric localization of calcium activity with dynamic microtubules. CaMKII (a kinase regulated by calcium) phosphorylates tau, a microtubule (MT) associated protein, thereby detaching tau from MTs and increasing MT dynamics. Thus tau may be one link between Wnt5a-evoked calcium signaling and increased MT dynamics which are required for growth cone turning in response to Wnt5a. Changes in MT distribution and dynamics will be imaged with TIRFM (total internal reflection fluorescence microscopy) in live growth cones following disruption of tau or overexpression of tau mutated at Ser 262 (the MT binding site) to determine if this site of tau is essential for axon outgrowth and guidance by Wnt5a. Since CaMKII phosphorylates tau at the Ser 262 MT binding site, these experiments have the potential to link asymmetry in MT dynamics to asymmetric calcium activity in growth cones evoked by Wnt5a gradients. In the third specific aim live cell imaging of fluorescently-labeled cytoskeletal elements, actin filaments and microtubules, will be carried out with TIRFM to understand how the cytoskeleton reorganizes in response to guidance cues to direct growth cone extension and turning behaviors. Manipulations of Wnt5a receptors and calcium signaling will be used to identify cytoskeletal changes associated with axon growth vs guidance. To determine whether these cytoskeletal changes also occur in an in vivo environment, confocal imaging of the cytoskeleton will be carried out in callosal growth cones extending in living cortical slices. PUBLIC HEALTH RELEVANCE: The goal of the proposed research is to characterize fundamental molecular mechanisms that regulate the growth of axons (nerve fibers) from the cerebral cortex and allow them to reach appropriate targets. An understanding of these developmental mechanisms will be essential for promoting regeneration of injured nerve fibers in the mammalian central nervous system.